Intravascular thrombosis is one of the most frequent pathological events and a major cause of morbidity and mortality. Critical steps in the development of acute coronary syndromes are the disruption, rupture, or erosion of artherosclerotic plaques with the formation of either partially or completely occlusive thrombus. Factors that stimulate thrombosis include vascular damage, stimulation of platelets, and activation of the coagulation cascade. Platelet adhesion to the exposed subendothelial surfaces of injured blood vessels, with subsequent platelet activation, and the resulting platelet-rich clot formation have been shown to be associated with various pathological conditions. The most prevalent vascular disease states are related to platelet dependent narrowing of the blood supply such as atherosclerosis and arteriosclerosis, acute myocardial infarction, chronic stable angina, unstable angina, transient ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, preeclampsia, embolism, restenosis or abrupt closure following angioplasty, carotid endarterectomy, anastomosis of vascular grafts, and etc. These conditions represent a variety of disorders thought to be initiated by platelet activation on vessel walls.
Platelets play a central role in blood clotting and blood vessel repair, rapidly adhering to sites of vessel damage where they undergo dramatic shape change to spread over the injury site. In addition to these physical changes during blood vessel injury, platelets also undergo a number of biochemical changes that must be tightly regulated. Regulation of platelets ensures that the formation of a blood clot is of sufficient size to seal off the damaged area, preventing blood loss, while not disrupting blood flow to vital organs by causing vessel occlusion.
Platelet aggregation refers to the adherence of platelets to each other, typically at the site of blood vessel damage. Clot retraction describes the contractile ability of platelets to consolidate or shrink the size of the blood clot once it has formed. This process is thought to be important for both maintenance of the vasculature and also the subsequent manner in which the blood clot is removed once wound healing has finished. Fibrinolysis, also known as clot lysis, refers to the process through which thrombi dissolve, as a consequence of activation of the fibrinolytic system.
Platelet aggregation, clot retraction, and fibrinolysis are important parts of thrombus regulation.
While clotting as a result of an injury to a blood vessel is a critical physiological process for mammals such as man, inappropriate clotting can also lead to disease states. For example, a pathological process called thrombosis results when platelet aggregation and/or a fibrin clot blocks (i.e., occludes) a blood vessel. Arterial thrombosis may result in ischemic necrosis of the tissue supplied by the artery. When the thrombosis occurs in a coronary artery, a myocardial infarction or heart attack can result. A thrombosis occurring in a vein may cause tissues drained by the vein to become edematous and inflamed. Thrombosis of a deep vein may be complicated by a pulmonary embolism. Preventing or treating clots in a blood vessel may be therapeutically useful by inhibiting formation of blood platelet aggregates, inhibiting formation of fibrin, inhibiting thrombus formation, inhibiting embolus formation, and for treating or preventing unstable angina, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation, ocular build up of fibrin, and reocclusion or restenosis of recanalized vessels.
Nitric oxide (NO) plays an important role during thrombus formation. During platelet aggregation and clot retraction, both inducible nitric oxide synthase (NOS) and constitutive nitric oxide synthase (eNOS) are transiently activated and then deactivated. The activity of nitric oxide (NO) as a vasodilator has been known for well over 100 years. (NO) is produced by NO synthases (NOS), which oxidize L-arginine to L-citrulline. It has recently become apparent that there are at least three types of NO synthase: (i) a constitutive, Ca++/calmodulin dependent enzyme, located in the endothelium, that releases NO in response to receptor or physical stimulation (eNOS); (ii) a constitutive, Ca++/calmodulin dependent enzyme, located in the brain, that releases NO in response to receptor or physical stimulation; and (iii) a Ca++ independent enzyme which is induced after activation of vascular smooth muscle, macrophages, endothelial cells, and a number of other cells by endotoxin and cytokines (NOS). All three NOS isoforms have a similar molecular structure and require multiple cofactors.
Given the role of NO during platelet aggregation, regulation of its synthesis has direct implications for platelet function during thrombus formation and acute coronary syndromes. More particularly, the ability to sustain NO production and release correlates with the inhibition of platelet aggregation and clot retraction.
For example, it has recently been shown that calpeptin, an inhibitor of the cellular protease calpain, can inhibit inducible NOS and inhibit platelet aggregation. Furthermore, U.S. patent application Ser. No. 09/953,590, filed Sep. 14, 2001 and published as US 2002/0128434A1, discloses that certain calpain inhibitors are useful as inhibitors against aggregation of platelets caused by thrombin. Similarly, inhibition of calpains for treating thrombosis or thrombotic platelet aggregation is described in U.S. patent application Ser. No. 09/847,872, filed May 2, 2001 and published as US 2002/0115665. U.S. Pat. No. 6,448,245, issued Sep. 10, 2002, provides methods and compounds for inhibiting calpains. However, while activity has focused on inducing nitric oxide synthase activity, it has not been previously known how to regulate constitutive endothelial nitric oxide synthase (eNOS) activity.
Integrin-mediated platelet adhesion triggers signal transduction cascades involving translocation of proteins and tyrosine phosphorylation events, ultimately causing large signaling complexes to be assembled. This activity is mediated by a number of platelet adhesive glycoproteins. The binding sites for fibrinogen, fibronectin, and other clotting factors have been located on the platelet membrane glycoprotein complex IIb/IIIa. When a platelet is activated by an agonist such as thrombin, the GPIIb/IIIa binding site becomes available to fibrinogen, eventually resulting in platelet aggregation and clot formation. Thus, the surface integrin GPIIb/IIIa (also known as the platelet integrin αIIbβ3) plays a key role during platelet aggregation.
A number of approaches have been taken to block platelet aggregation by disrupting the binding of fibrinogen to its receptor, IIb/IIIa. Examples of agents that inhibit their interaction include for example various benzoic acid and phenylacetic acid (see U.S. Pat. No. 5,039,805); seven membered ring containing bicyclic compounds (see PCT International patent application WO 93/00095); bicyclic compounds having fused six membered rings (quinazoline-3-alkanoic acid derivates) (see EP 456835); nonpeptidyl integrin inhibitors which are bicyclic 6 and 7 membered fused ring systems (see PCT International patent application WO 93/08174); 6,5-bicyclic compounds (Patent Application WO94/12478 and Patent Application WO94/08962); novel oxoquinazolin derivatives (see British Patent application GB 2276384). Furthermore, the design of non-peptidal inhibitors of fibrinogen-glycoprotein IIb/IIIa binding has been described (see McDowell, et. al., J. Am. Chem. Soc. 1994, 116, pp. 5069-5076 and Blackburn and Gadek, “Chapter 9. Glycoprotein IIb/IIIa Antagonists”, Annual Reports in Medicinal Chemistry—28, Section II—Cardiovascular and Pulmonary Agents, pp 79-88, 1993, publ. by Academic Pres, Inc.).
One signaling molecule activated during platelet activation is the cellular kinase syk. Maguire et al., Proteomics 2:642-8 (2002) discloses the identification of syk as one of several phosphotyrosine proteins present in thrombin-activated platelets. syk can be immunoprecipitated from platelets stimulated by von Willebrand factor and ristocetin, modeling platelet adhesion and thrombus formation [Goto et al., Circulation 106:266-72 (2002)]. For example, syk associates directly with the integrin αIIbβ3 in platelets, demonstrating that αIIbβ3-associated syk is activated during platelet stimulation [Sarkar et al., Biochem. J. 338:677-80 (1999)].
Anti-thrombotic agents can block or inhibit thrombus formation, as discussed above; however, they are not very effective in dissolving a pre-formed thrombus or to help in fibrinolysis. Thus, terminal thrombus formation may cause myocardial infarction and/or ischemic chest pain. Instead, the current treatment for total blockage by thrombus formation is either angio-balloon-plasty and/or bypass surgery.
A number of agents which promote fibrinolysis after a thrombus has been formed have been identified. Fibrinolysis is promoted by the conversion of plasminogen to plasmin. The most important agents are the physiologic plasminogen activators tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA and u-PA are serine proteinases that activate the proenzyme plasminogen to the broad specificity enzyme plasmin. [Collen and Lijnen, Blood 78:3114 (1991)]
However, fibrinolytic agents typically have problems because of the inhibitory effect of platelets on clot lysis. Activated platelets at sites of thrombus secrete agents which inhibit proteolytic processing of plasminogen to active plasmin. The serpin plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of both t-PA and u-PA, and constitutes a critical regulator of plasminogen activation. [Sprengers and Kluft, Blood:69:381 (1987)] Several animal and clinical studies have associated elevations in plasma PAI-1 with increased risk for thrombosis, whereas a drop in plasma PAI-1 levels may be a cause of recurrent bleeding.
During fibrinolysis, plasmin is very rapidly and specifically inhibited by alpha2-antiplasmin (α2-AP), which circulates in plasma at a high concentration of 1 μmol/L. Whereas α2-AP is synthesized in the liver and released into the circulation, the origin of active PAI-1 in the circulation is less well-defined. PAI-1 is produced by several cell types, including endothelial cells, smooth muscle cells, fibroblasts, and hepatocytes. Two distinct pools of PAI-1 exist in the circulation, one in platelets and one in plasma. Human platelets are a major reservoir of PAI-1, with up to 90% of the circulating human PAI-1 contained within platelet α-granules, yielding up to 700 ng PAI-1 per 109 platelets. However, platelet PAI-1 exists predominantly in a latent or inactive form, suggesting its effect on fibrinolysis to be rather limited. Nevertheless, the inhibitory effect of platelets on clot lysis was proposed to be mediated partly by platelet PAI-1, a conclusion supported by differential clot lysis efficiency in the presence of normal platelets or platelets derived from PAI-1-deficient patients.
There is a need in the area of cardiovascular and cerebrovascular therapeutics for alternative agents which can be used in the prevention and treatment of thrombi. Accordingly, it would be desirable to have improved methods for treating thrombosis. More particularly, it would be desirable to have improved compounds to inhibit platelet aggregation and clot retraction, and promote fibrinolysis. There is also a need to have better assays for screening for such compounds.